In this renewal application, we describe a new pathway signaling neutrophil (PMN) influx and damage to the airways that was delineated in the parent grant and that may play a role as an initiator or cofactor for chronic lung diseases such as obstructive pulmonary disease (COPD), cystic fibrosis (CF), and chronic lung transplant rejection. Specifically, chemical or enzymatic breakdown of collagen releases a tripeptide, PGP, and/or a related PGP-containing sequence that is chemotactic for PMN in vitro. We demonstrate that introduction of PGP into the airways causes a robust influx of PMN, but not monocytes. Remarkably, the PMN chemotactic activity of PGP may be due to a marked structural relatedness to a receptor binding domain of ELR+ CXC chemokines such as IL-8 which contain this collagen sequence or a close analog. Prolonged airway exposure to this peptide causes alveolar enlargement and right ventricular hypertrophy and thus recapitulates aspects of COPD. We have developed a potent antagonist, Arg-Thr-Arg, termed RTR, which binds PGP in solution and blocks its in vitro chemotactic activity and in vivo pathophysiologic effects. Likely as a result of shared structure with PGP, RTR also blocks IL-8 activity. In a mouse model of LPS-induced COPD, RTR completely inhibited alveolar enlargement and right ventricular hypertrophy. The degradation of collagen and release of PGP is shown to be a stepwise process initially involving matrix metalloproteases (MMP)-8 and/or 9 with prolyl endopeptidase (PE) catalyzing the final reaction. We have found that a highly specific PE inhibitor, N-benzyloxycarbonyl (Z) prolyl prolinal (ZPP), can block airway PGP production as well as PMN influx in response to LPS. Surprisingly, the very potent blunting of PMN influx by ZPP appears to be due not only to PE inhibition but also as a result of ZPP antagonism of CXCR 1/2 likely as a result of structural similarity between ZPP and PGP. This suggests the intriguing possibility, to be explored in this proposal, that a single compound can be developed that is both a protease inhibitor and receptor antagonist. If successful, this would represent an entirely new chimeric type of lead compound for the treatment of neutrophilic inflammation, in general, and lung diseases in particular. We have found that PGP is present in bronchoalveolar lavage fluids (BALF) and/or sputum from virtually all CF, COPD or chronic lung transplant rejection patients but not controls or asthmatics. Furthermore, sputum from COPD and CF patients but not control individuals contains all the enzymatic machinery necessary for the ex vivo generation of PGP from purified collagen and such PGP production can be blocked by the PE inhibitor, ZPP. Collectively, these findings lead us to hypothesize that PGP and PE represent novel biomarkers for COPD that may contribute to disease and that PGP and PE represent attractive therapeutic targets at the level of the ligand, receptor, and generating enzyme. PUBLIC HEALTH RELEVANCE: COPD is now the fourth leading cause of death in the U.S. and is expected to be the third by 2020. This disease causes much human suffering as well as a large monetary burden. An understanding of COPD that leads to successful therapeutics has been hampered by a lack of biomarkers to diagnose and use as endpoints in clinical trials as well as a lack of a fundamental understanding of the disease process itself. If successful, the current proposal will yield fundamental insights into the disease process and a new and specific biomarker for COPD as well as suggesting new therapeutic targets.